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Developing Different Formats for Combining Aqueous Two-Phase Systems with Isothermal DNA Amplification


Infectious diseases remain a public health concern worldwide. They severely impact resource-poor areas, where the top 10 causes of deaths include diarrhoeal diseases, HIV/AIDS, and tuberculosis. One gold-standard detection method for these infectious agents is nucleic acid amplification with the polymerase chain reaction (PCR). Unfortunately, its requirements for laboratory trained personnel and equipment prevent it from becoming a useful diagnostic tool at the point of care. To eliminate the need for a thermocycler, isothermal DNA amplification techniques were developed, but they cannot stand alone as complete diagnostic tools. They still require an extensive DNA preparation step prior to amplification. In addition, before it can be brought to an end-user, the entire detection scheme from sample preparation to end detection needs to be incorporated on a platform, with all of the reagents stored.

This thesis tackles the first aim by describing the development of an integrated method to extract, purify, and amplify DNA in one step using a micellar aqueous two-phase system (ATPS) with thermophilic helicase-dependent amplification (tHDA). This one-pot system was able to detect a target sequence from whole bacteria samples with cell concentrations as low as 10^2 colony forming units/mL. This is the first known application of an ATPS to isothermal DNA amplification.

For the second aim, this thesis describes the work that has been done so far as part of the initial step to the development of a fully-integrated nucleic acid testing device using a microfluidic platform. To avoid relying on the cleanroom for fabrication and external pumps for fluid flow in the chips, vacuum-driven chips were made from 3D-printed master molds for the first time. In addition, to our knowledge, we demonstrated the first dehydration of a full reaction set up for the recombinase polymerase amplification (RPA), including all of the enzymes and the primers but not the reaction activator magnesium acetate. The promising results will serve as a good starting point for our work in developing stand-alone diagnostic devices that can be used at the point of care and allow for a fully automated sample-to-result testing procedure.

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